EP3608452A1 - Method for constructing amplicon library through one-step process - Google Patents

Method for constructing amplicon library through one-step process Download PDF

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EP3608452A1
EP3608452A1 EP18780976.9A EP18780976A EP3608452A1 EP 3608452 A1 EP3608452 A1 EP 3608452A1 EP 18780976 A EP18780976 A EP 18780976A EP 3608452 A1 EP3608452 A1 EP 3608452A1
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amplicon
seq
gene
sequence
designed according
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French (fr)
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EP3608452A4 (en
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Hai Yan
Sizhen WANG
Yuchen JIAO
Dayong Xu
Qiaosong ZHENG
Xiao Shi
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Genetron Health Beijing Co Ltd
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Genetron Health Beijing Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/191—Modifications characterised by incorporating an adaptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/122—Massive parallel sequencing

Definitions

  • the invention relates to a field of biotechnology, and in particular to a method for rapidly constructing amplicon library through one-step process.
  • NGS Next-generation sequencing
  • the traditional method for constructing an amplicon library in the Life NGS platform is cumbersome, requires PCR amplification, digestion, addition, and purification, and takes about 5 hours. Further, because of the need to open the lid in a multi-step operation, the library is easily contaminated and the library loss rate is high.
  • the cost of establishing a library for a single sample is relatively high, which is about 200-1000 RMB per case.
  • the object of the present invention is to provide a method for rapidly constructing an amplicon library through one-step process.
  • the method can construct the amplicon library by one-step PCR in a simple and rapid manner, and since the barcode is introduced before the start of PCR, the possibility of cross-contamination between samples and libraries is greatly reduced, and the requirements of the experimental site partition can be simplified.
  • the method also controls the cost of establishing a single sample library at 30 RMB percase.
  • the present invention provides a method for constructing an amplicon library of a DNA sample, comprising the following steps.
  • the first linker sequence comprises a sequence of SEQ ID: 1, and the nucleotide sequence of the sequence of SEQ ID: 1 is GGCATACGTCCTCGTCTA.
  • the second linker sequence comprises a sequence of SEQ ID: 2, and the nucleotide sequence of the sequence of SEQ ID: 2 is TCTATGGGCAGTCGGTGAT.
  • the third linker sequence comprises a sequence of SEQ ID:3, and a nucleotide sequence of the sequence of SEQ ID:3 is CCATCTCATCCCTGCGTGTCTCCGACTCAG.
  • the universal sequence comprises a sequence of SEQ ID: 4, and a nucleotide sequence of the sequence of SEQ ID: 4 is CCACTACGCCTCCGCTTTCCTC.
  • the barcode sequence in the upstream universal primer is the same.
  • the barcode sequences in the upstream universal primers are different.
  • the barcode sequence corresponds to the sample.
  • the barcode sequence is different between different samples. As long as different samples can be distinguished, the barcode sequence is not specific and its sequence can be changed.
  • the concentration of the upstream fusion primer designed according to any one of the target amplicon, the concentration of downstream fusion primer designed according to any one of the target amplicon, the concentration of upstream universal primer, and the concentration of downstream universal primer are all 100 ⁇ M.
  • the upstream fusion primer designed according to a target amplicon is a combination of upstream fusion primers designed according to each target amplicon
  • the downstream fusion primer designed according to the target amplicon is a combination of downstream fusion primers designed according to each target amplicon
  • the molar ratio of the upstream fusion primer designed according to any one of the target amplicon to the downstream fusion primer designed according to the target amplicon is 1:1; the molar ratio of the upstream universal primer to the downstream universal primer is 1:1.
  • the specific amount of upstream universal primers and downstream universal primers should be adjusted according to the number of target amplicons during PCR amplification. For example, when PCR amplification, 5 target amplicons need to be amplified and 22 target amplicons need to be amplified, the specific amount of the upstream universal primer and the downstream universal primer may be different, and a specific amount of the upstream universal primer and the downstream universal primer may be determined by those skilled in the art according to conventional techniques in the art.
  • the DNA sample is genomic DNA.
  • the genomic DNA is extracted from a tissue sample or a formalin-fixed paraffin-embedded sample.
  • the target amplicon comprises at least one selected from the group consisting of 22 target amplicons:
  • the specific upstream primer sequence designed according to the Chr2:29432588-29432707 (Hg19) amplicon of the ALK gene is shown as SEQ ID: 27: ACTGCCTCTTGACCTGTCC;
  • the specific downstream primer sequence designed according to the Chr2:29432588-29432707 (Hg19) amplicon of the ALK gene is shown as SEQ ID: 28: TAAGGGACAAGCAGCCACAC.
  • the specific upstream primer sequence designed according to the Chr2: 29443616-29443730 (Hg19) amplicon of ALK gene is shown as SEQ ID: 29: CCAGACTCAGCTCAGTTAATTTTGG; the specific downstream primer sequence designed according to the Chr2: 29443616-29443730 (Hg19) amplicon of the ALK gene is shown as SEQ ID: 30: CGGAGGAAGGACTTGAGGT.
  • the specific upstream primer sequence designed according to the Chr7: 140453091-140453197(Hg19) amplicon of BRAF gene is shown as SEQ ID: 31: CTACTGTTTTCCTTTACTTACTACACCTC; the specific downstream primer sequence designed according to the Chr7: 140453091-140453197(Hg19) amplicon of the BRAF gene is shown as SEQ ID: 32: CCTCAATTCTTACCATCCACAAAATGG.
  • the specific upstream primer sequence designed according to the Chr7: 55241604-55241726(Hg19) amplicon of EGFR gene is shown as SEQ ID: 33: TGACCCTTGTCTCTGTGTTCTTG; the specific downstream primer sequence designed according to the Chr7: 55241604-55241726(Hg19) amplicon of the BRAF gene is shown as SEQ ID: 34: CCAGGGACCTTACCTTATACACC.
  • the specific upstream primer sequence designed according to the Chr7: 55242398-55242513 (Hg19) amplicon of EGFR gene is shown as SEQ ID:35: ACAATTGCCAGTTAACGTCTTCC; the specific downstream primer sequence designed according to the Chr7: 55242398-55242513 (Hg19) amplicon of the EGFR gene is shown as SEQ ID: 36: ACACAGCAAAGCAGAAACTCAC.
  • the specific upstream primer sequence designed according to the Chr7: 55248970-55249096 (Hg19) amplicon of EGFR gene is shown as SEQ ID: 37: GAAGCCACACTGACGTGC; the specific downstream primer sequence designed according to the Chr7: 55248970-55249096 (Hg19) amplicon of the EGFR gene is shown as SEQ ID: 38: GTGTTCCCGGACATAGTCCAG.
  • the specific upstream primer sequence designed according to the Chr7: 55259505-55259621 (Hg19) amplicon of EGFR gene is shown as SEQ ID: 39: CCGCAGCATGTCAAGATCACA; the specific downstream primer sequence designed according to the Chr7: 55259505-55259621 (Hg19) amplicon of the EGFR gene is shown as SEQ ID: 40: TAAACAATACAGCTAGTGGGAAGGC.
  • the specific upstream primer sequence designed according to the Chr17: 37880969-37881082 (Hg19) amplicon of ERBB2 gene is shown as SEQ ID: 41: CATACCCTCTCAGCGTACCC; the specific downstream primer sequence designed according to the Chr17: 37880969-37881082 (Hg19) amplicon of the ERBB2 gene is shown as SEQ ID: 42: CGGACATGGTCTAAGAGGCAG.
  • the specific upstream primer sequence designed according to the Chr12: 25380261-25380363 (Hg19) amplicon of KRAS gene is shown as SEQ ID: 43: TGCACTGTAATAATCCAGACTGTGT; the specific downstream primer sequence designed according to the Chr12: 25380261-25380363 (Hg19) amplicon of the KRAS gene is shown as SEQ ID: 44: AGTCCTCATGTACTGGTCCCTC.
  • the specific upstream primer sequence designed according to the Chr12: 25398183-25398310 (Hg19) amplicon of KRAS gene is shown as SEQ ID: 45: AAGGCCTGCTGAAAATGACTGA; the specific downstream primer sequence designed according to the Chr12: 25398183-25398310 (Hg19) amplicon of the KRAS gene is shown as SEQ ID: 46: AAAGAATGGTCCTGCACCAGTA.
  • the specific upstream primer sequence designed according to the Chr7: 116340233-116340335 (Hg19) amplicon of MET gene is shown as SEQ ID: 47: TCGATCTGCCATGTGTGCATT;
  • the specific downstream primer sequence designed according to the Chr7: 116340233-116340335 (Hg19) amplicon of the MET gene is shown as SEQ ID: 48: GGGAACTGATGTGACTTACCCT.
  • the specific upstream primer sequence designed according to the Chr7: 116411880-116412005 (Hg19) amplicon of MET gene is shown as SEQ ID: 49: CCATGATAGCCGTCTTTAACAAGC; the specific downstream primer sequence designed according to the Chr7: 116411880-116412005 (Hg19) amplicon of the MET gene is shown as SEQ ID: 50: AGCTCGGTAGTCTACAGATTCATTT.
  • the specific upstream primer sequence designed according to the Chr7: 116417426-116417546 (Hg19) amplicon of MET gene is shown as SEQ ID: 51: ATGTTACGCAGTGCTAACCAAG; the specific downstream primer sequence designed according to the Chr7: 116417426-116417546 (Hg19) amplicon of the MET gene is shown as SEQ ID: 52: GTTGCAAACCACAAAAGTATACTCCA.
  • the specific upstream primer sequence designed according to the Chr7: 116423399-116423499 (Hg19) amplicon of MET gene is shown as SEQ ID: 53: CAGTCAAGGTTGCTGATTTTGGTC; the specific downstream primer sequence designed according to the Chr7: 116423399-116423499 (Hg19) amplicon of the MET gene is shown as SEQ ID: 54: CACATCTGACTTGGTGGTAAACTT.
  • the specific upstream primer sequence designed according to the Chr1: 115256507-115256586 (Hg19) amplicon of NRAS gene is shown as SEQ ID: 55: CACCCCCAGGATTCTTACAGAAAA; the specific downstream primer sequence designed according to the Chr1: 115256507-115256586 (Hg19) amplicon of the NRAS gene is shown as SEQ ID: 56: TTCGCCTGTCCTCATGTATTGG.
  • the specific upstream primer sequence designed according to the Chr1: 115258651-115258755 (Hg19) amplicon of NRAS gene is shown as SEQ ID: 57: CTGAGTACAAACTGGTGGTGGT; the specific downstream primer sequence designed according to the Chr1: 115258651-115258755 (Hg19) amplicon of the NRAS gene is shown as SEQ ID: 58: TGAGAGACAGGATCAGGTCAGC.
  • the specific upstream primer sequence designed according to the Chr3: 178936056-178936179 (Hg19) amplicon of PIK3CA gene is shown as SEQ ID: 59: GGAAAATGACAAAGAACAGCTCAAAG; the specific downstream primer sequence designed according to the Chr3: 178936056-178936179 (Hg19) amplicon of the PIK3CA gene is shown as SEQ ID: 60: AACATGCTGAGATCAGCCAAATTC.
  • the specific upstream primer sequence designed according to the Chr3: 178952000-178952092 (Hg19) amplicon of PIK3CA gene is shown as SEQ ID: 61: ATGCCAGAACTACAATCTTTTGATGAC; the specific downstream primer sequence designed according to the Chr3: 178952000-178952092 (Hg19) amplicon of the PIK3CA gene is shown as SEQ ID: 62: CAATCCATTTTTGTTGTCCAGCC.
  • the specific upstream primer sequence designed according to the Chr17: 7577027-7577154 (Hg19) amplicon of TP53 gene is shown as SEQ ID: 63: CTCTTTTCCTATCCTGAGTAGTGGTAATC; the specific downstream primer sequence designed according to the Chr17: 7577027-7577154 (Hg19) amplicon of the TP53 gene is shown as SEQ ID: 64: CTTCTTGTCCTGCTTGCTTACC.
  • the specific upstream primer sequence designed according to the Chr17: 7577507-7577613 (Hg19) amplicon of TP53 gene is shown as SEQ ID: 65: TCTTGGGCCTGTGTTATCTCCTAG; the specific downstream primer sequence designed according to the Chr17: 7577507-7577613 (Hg19) amplicon of the TP53 gene is shown as SEQ ID: 66: GCAAGTGGCTCCTGACCTG.
  • the specific upstream primer sequence designed according to the Chr17: 7578182-7578298 (Hg19) amplicon of TP53 gene is shown as SEQ ID: 67: CCTCTGATTCCTCACTGATTGCTC; the specific downstream primer sequence designed according to the Chr17: 7578182-7578298 (Hg19) amplicon of the TP53 gene is shown as SEQ ID: 68: CCCCAGTTGCAAACCAGAC.
  • the specific upstream primer sequence designed according to the Chr17: 7578389-7578537 (Hg19) amplicon of TP53 gene is shown as SEQ ID: 69: CAGTACTCCCCTGCCCTCAA; the specific downstream primer sequence designed according to the Chr17: 7578389-7578537 (Hg19) amplicon of the TP53 gene is shown as SEQ ID: 70: ACCATCGCTATCTGAGCAGC.
  • the target amplicons are the following 22 species:
  • the molar ratio of the combination of the upstream fusion primers designed according to the above 22 target amplicons, the combination of the downstream fusion primers designed according to the above 22 target amplicons, the upstream universal primer and the downstream universal primer is: 0.1-0.3: 0.1-0.3: 0.5-1: 0.5-1, for example, 0.1:0.1:0.5:0.5.
  • the PCR reaction system includes the following components: PCR master mix 10 ⁇ l; DNA sample 1-8 ⁇ l total 20 ng; Primer combination for constructing an amplicon library of the same DNA sample 2 ⁇ l; DNAase-free H2O making up to 20 ⁇ l.
  • the PCR master mix is KAPA HiFi PCR Kits 2 ⁇ .
  • the reaction procedure for performing PCR is: Temperature Time Number of cycles 98°C 30s 98°C 10s 22 cycles 60°C 90s 72°C 90s 72°C 10min 4°C ----
  • a step of purifying the PCR amplification product is also included.
  • the present invention has the following advantages:
  • the method disclosed in the present invention is based on the design of the PGM platform, and can effectively amplify multiple target regions (amplicons) at the same time.
  • the present invention only involves one round of PCR reaction and one round of product purification steps, which greatly simplifies the experimental operation of the existing commercial kit (such as PCR process, purification step, digestion and joints, etc.) Step), and saves the construction time.
  • the entire database construction process only takes 2.5 hours (including the same sample of DNA and RNA database).
  • Streamlined bioinformatics analysis process is obtained.
  • the amplicon library obtained by the method has a single structure and reliable data, and the DNA strand composition of the obtained library is simple and clear, and the subsequent bioinformatics analysis is more simplified.
  • the library is only needed to be quantified by the instrument "Qubit 2.0", which eliminates quantification step by the instrument "qPCR”. Therefore, the database construction time is shortened and corresponding operation steps are reduced, and the experimental errors that may be caused by the cumbersome experimental process are avoided.
  • the samples to be tested are 6 FFPE samples (ie, formalin-fixed paraffin-embedded samples, FFPE stands for Formalin-Fixed and Parrffin-Embedded), 4 of which are FFPE samples from patients with non-small cell lung cancer, and 2 are non- FFPE samples from patients with cancer.
  • Amplified primers are used to construct an amplicon DNA library from 6 FFPE samples using a specific designed fusion primer. The specific process is as follows:
  • the genomic DNA in the FFPE sample is extracted using the "Qiagen FFPE DNA Kit".
  • the detailed steps for extraction can be referred to the kit instructions.
  • the genomic DNA is dissolved in "Tris-HCl” buffer, quality of the extracted DNA is detected using "Nano Drop”. After the concentration of the sample DNA is detected using the instrument "quiz 3.0", each genomic DNA sample is diluted to a concentration of 20 ng/ ⁇ l.
  • An upstream fusion primer is designed according to the target amplicon.
  • the upstream fusion primer includes a first linker sequence arranged in the order of 5' to 3' and a specific upstream primer sequence that is designed according to the target amplicon;
  • a downstream fusion primer is designed according to the target amplicon.
  • the downstream fusion primer includes a second linker sequence arranged in the order of 5' to 3' and a specific downstream primer sequence designed according to the target amplicon;
  • An upstream universal primer comprises a third linker sequence, barcode sequence and a first linker sequence arranged in the order of 5' to 3'; and
  • a downstream universal primer comprises a universal sequence and a second linker sequence arranged in the order of 5' to 3'.
  • the information of the specific upstream primer sequence and the specific downstream primer sequence designed according to the target amplicon are as follows: Information on the different target amplicons is given in the table below, and the specific upstream primer sequence "Special Primer Start” and the specific downstream primer sequence "Special Primer End” designed for these amplicons are also given. Sequences of upstream fusion primers designed according to the target amplicon, downstream fusion primers designed according to the target amplicon, upstream universal primers, and downstream universal primers are also given. Puf representes an alternative upstream universal primer and Pur representes a downstream universal primer.
  • the first linker sequence is GGCATACGTCCTCGTCTA
  • the second linker sequence is TCTATGGGCAGTCGGTGAT
  • the third linker sequence is CCATCTCATCCCTGCGTGTCTCCGACTCAG
  • the universal sequence is CCACTACGCCTCCGCTTTCCTC.
  • the specific PCR reaction system is as follows:
  • Primer combinations for constructing an amplicon library of the same DNA sample are prepared by the following methods: (1) the upstream universal primer, the downstream universal primer, and each upstream fusion primer designed according to the 22 target amplicons and each downstream fusion primer are dissolved in water to a concentration of 100 ⁇ M; (2) 22 upstream fusion primers with a serial number ranging from small to large are respectively mixed with a concentration of 100 ⁇ M, and the molar ratio is 1:2:1:4:2:1:2:4:2:2:2:2:2:4:2:2:2:4:2:4:2:4:2:2, so as to obtain the upstream fusion primer combination, and 22 downstream fusion primers with a concentration of 100 ⁇ M are respectively mixed with the corresponding upstream fusion primers in equal volume to obtain a downstream fusion primer combination, and then the upstream fusion primer combination and the downstream fusion primer combination are mixed in equal volume; (3) mixing in equal volume of upstream universal primers and downstream universal primers with concentrations of 100 ⁇
  • the PCR instrument is the 2720 Thermal Cycler of Applied Bio-system.
  • the PCR reaction procedure is as follows:
  • Figure 1 shows the distribution of amplified products detected by Agilent 2200 TapeStation Systems after the completion of the library.
  • the abscissa is the length of the fragment
  • the ordinate is the signal intensity (FU)
  • the lower peak is the 25 bp position marker
  • the upper peak is a 1500 bp position marker.
  • the PCR products obtained by PCR amplification are concentrated in the range of 241-271 bp.
  • Figure 1 shows that the experimental results are consistent with the experimental design. From Figure 1 , the size of the constructed library and the library concentration can be judged.
  • the amplicon library is obtained by the fusion primer one-step method.
  • the amplicon sequencing is performed using the chip 318 of the Ion PGM platform, and the data amount of each library is 50 M bps.
  • the average sequencing depth of each sample is not less than 1600X, and the single amplicon sequencing depth reached 600X.
  • the obtained sequencing results are shown in Fig. 2 . From Fig. 2 , it is possible to further analyze whether or not each amplicon of the 22 amplicons is amplified and the amplification uniformity of each amplicon.
  • the results of sequencing are analyzed by data processing and bioinformatics analysis to obtain mutations in the detected genes.
  • the data processing process includes conversion, quality control and sequence alignment of the sequencing data (reference genome is NCBI GRCh37/Hg19), mutation site analysis and other processes, and the mutation information of the detected samples is obtained through data processing analysis.
  • the actual sample collection is as follows: Among the FFPE samples of 6 subjects, no tumor-related mutations are detected in 2 normal human samples, among the 4 FFPE samples of tumor patients, p.R248W mutation is detected in Sample1, p.T790M mutation is detected in sample2, p.G12A mutation is detected in sample3, and p.E545K mutation is detected in Sample4. This result is consistent with the results of the sanger test. The practical applicability and good specificity of the present invention are fully illustrated.

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Abstract

The present invention discloses a method for rapidly constructing amplicon library through one-step process, comprising the following steps: 1. Synthesizing a primer combination for constructing an amplicon library of a DNA sample, the primer combination of the amplicon library used to construct the DNA sample includes: an upstream fusion primer designed according to the target amplicon, a downstream fusion primer designed according to the target amplicon, an upstream universal primer and a downstream universal primer; 2. Constructing a PCR reaction system for the DNA sample; 3. Performing PCR. This method according to the present invention can be used to construct an amplicon library in a simple and rapid manner, and since the barcode is introduced before the start of PCR, the possibility of cross-contamination between the sample and the library is greatly reduced.

Description

  • The present invention claims priority of a Chinese patent application filed with the China Patent Office by the Genetron Health(Beijing) Co., Ltd. on April 5, 2017, with the application number of 201710218529.4 , and entitled "method for rapidly constructing amplicon library through one-step process". The entire content of this application are incorporated herein by reference.
    • TECHNICAL FIELD
  • The invention relates to a field of biotechnology, and in particular to a method for rapidly constructing amplicon library through one-step process.
    • BACKGROUND OF RELATED ART
  • Next-generation sequencing (NGS) has been widely used in disease research, diagnosis and treatment in recent years due to its high throughput, high sensitivity, and high automation. Compared with traditional detection method, NGS technology can achieve multi-gene parallel detection and save samples. Besides, it has higher sensitivity which can restore the panoramic view of tumor variation in a more realistic way. However, the traditional method for constructing an amplicon library in the Life NGS platform is cumbersome, requires PCR amplification, digestion, addition, and purification, and takes about 5 hours. Further, because of the need to open the lid in a multi-step operation, the library is easily contaminated and the library loss rate is high. In addition, in the traditional method of constructing the amplicon library, the cost of establishing a library for a single sample is relatively high, which is about 200-1000 RMB per case.
  • The information disclosed in background part is only intended to enhance an understanding of the general background of the invention, and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
    • SUMMARY OF THE INVENTION
  • The object of the present invention is to provide a method for rapidly constructing an amplicon library through one-step process. The method can construct the amplicon library by one-step PCR in a simple and rapid manner, and since the barcode is introduced before the start of PCR, the possibility of cross-contamination between samples and libraries is greatly reduced, and the requirements of the experimental site partition can be simplified. The method also controls the cost of establishing a single sample library at 30 RMB percase.
  • To achieve the above object, the present invention provides a method for constructing an amplicon library of a DNA sample, comprising the following steps.
    • Step 1: synthesizing a primer combination for constructing an amplicon library of a DNA sample, the primer combination of the amplicon library that is used to construct the DNA sample includes:
      • a fusion primer that is designed according to a target amplicon. The upstream fusion primer comprises a first linker sequence (Bridge sequence) arranged in the order of 5' to 3' and a specific upstream primer sequence designed according to the target amplicon;
      • a downstream fusion primer that is designed according to the target amplicon. The downstream fusion primer comprises a second linker sequence (trP1 sequence) arranged in the order of 5' to 3' and a specific downstream primer sequence designed according to the target amplicon;
      • an upstream universal primer which comprises a third linker sequence (A sequence), a barcode sequence and a first linker sequence arranged in the order of 5' to 3'; and
      • a downstream universal primer which comprises a universal sequence (Uni sequence) and a second linker sequence arranged in the order of 5' to 3';
    • Step 2: constructing a PCR reaction system for the DNA sample, and mixing the upstream fusion primers designed according to the target amplicon, the downstream fusion primers designed according to the target amplicon, the upstream universal primers and the downstream universal primers together, to serve as a primer combination in the PCR reaction system;
    • Step 3: performing PCR.
  • In an embodiment of the present invention, the first linker sequence comprises a sequence of SEQ ID: 1, and the nucleotide sequence of the sequence of SEQ ID: 1 is GGCATACGTCCTCGTCTA.
  • In an embodiment of the present invention, the second linker sequence comprises a sequence of SEQ ID: 2, and the nucleotide sequence of the sequence of SEQ ID: 2 is TCTATGGGCAGTCGGTGAT.
  • In an embodiment of the present invention, the third linker sequence comprises a sequence of SEQ ID:3, and a nucleotide sequence of the sequence of SEQ ID:3 is CCATCTCATCCCTGCGTGTCTCCGACTCAG.
  • In an embodiment of the present invention, the universal sequence comprises a sequence of SEQ ID: 4, and a nucleotide sequence of the sequence of SEQ ID: 4 is CCACTACGCCTCCGCTTTCCTC.
  • In an embodiment of the present invention, in the primer combination for constructing an amplicon library of the same DNA sample, the barcode sequence in the upstream universal primer is the same. In the primer combinations for constructing amplicon libraries of the different DNA samples, the barcode sequences in the upstream universal primers are different. The barcode sequence corresponds to the sample. The barcode sequence is different between different samples. As long as different samples can be distinguished, the barcode sequence is not specific and its sequence can be changed.
  • In an embodiment of the present invention, the concentration of the upstream fusion primer designed according to any one of the target amplicon, the concentration of downstream fusion primer designed according to any one of the target amplicon, the concentration of upstream universal primer, and the concentration of downstream universal primer are all 100 µM.
  • In an embodiment of the present invention, when the number of target amplicons in the same PCR reaction is greater than 1, the upstream fusion primer designed according to a target amplicon is a combination of upstream fusion primers designed according to each target amplicon, the downstream fusion primer designed according to the target amplicon is a combination of downstream fusion primers designed according to each target amplicon.
  • In an embodiment of the present invention, the molar ratio of the upstream fusion primer designed according to any one of the target amplicon to the downstream fusion primer designed according to the target amplicon is 1:1; the molar ratio of the upstream universal primer to the downstream universal primer is 1:1. The specific amount of upstream universal primers and downstream universal primers should be adjusted according to the number of target amplicons during PCR amplification. For example, when PCR amplification, 5 target amplicons need to be amplified and 22 target amplicons need to be amplified, the specific amount of the upstream universal primer and the downstream universal primer may be different, and a specific amount of the upstream universal primer and the downstream universal primer may be determined by those skilled in the art according to conventional techniques in the art.
  • In an embodiment of the present invention, the DNA sample is genomic DNA.
  • In an embodiment of the present invention, the genomic DNA is extracted from a tissue sample or a formalin-fixed paraffin-embedded sample.
  • In an embodiment of the present invention, the target amplicon comprises at least one selected from the group consisting of 22 target amplicons:
    • Chr2:29432588-29432707 (Hg19) amplicon of the ALK gene, the sequence of which is shown in SEQ ID:5:TAAGGGACAAGCAGCCACACCCCATTCTTGAGGGGCTGAGGTGGAAGAGACAGGCCCGGA GGGGTGAGGCAGTCTTTACTCACCTGTAGATGTCTCGGGCCATCCCGAAGTCTCCAATCTTGGCC ACTCTTCCAGGGCCTGGACAGGTCAAGAGGCAGT;
    • Chr2: 29443616-29443730 (Hg19) amplicon of the ALK gene, the sequence of which is shown in SEQ ID:6:CGGAGGAAGGACTTGAGGTCTCCCCCCGCCATGAGCTCCAGCAGGATGAACCGGGGCAGGG ATTGCAGGCTCACCCCAATGCAGCGAACAATGTTCTGGTGGTTGAATTTGCTGCAGAGCAGAGAG GGATGTAACCAAAATTAACTGAGCTGAGTCTGG;
    • Chr7: 140453091-140453197(Hg19) amplicon of the BRAF gene, the sequence of which is shown in SEQ ID:7:CCTCAATTCTTACCATCCACAAAATGGATCCAGACAACTGTTCAAACTGATGGGACCCACTC CATCGAGATTTCACTGTAGCTAGACCAAAATCACCTATTTTTACTGTGAGGTCTTCATGAAGAAA TATATCTGAGGTGTAGTAAGTAAAGGAAAACAGTAG;
    • Chr7: 55241604-55241726(Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:8: TGACCCTTGTCTCTGTGTTCTTGTCCCCCCCAGCTTGTGGAGCCTCTTACACCCAGTGGAGAAGCT CCCAACCAAGCTCTCTTGAGGATCTTGAAGGAAACTGAATTCAAAAAGATCAAAGTGCTGGGCT CCGGTGCGTTCGGCACGGTGTATAAGGTAAGGTCCCTGG;
    • Chr7: 55242398-55242513 (Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:9: ACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGA AAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGGA AATCCTCGATGTGAGTTTCTGCTTTGCTGTGT;
    • Chr7: 55248970-55249096 (Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:10:GAAGCCACACTGACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGAC AACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCT CATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACAC;
    • Chr7: 55259505-55259621 (Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:11:CCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAA GAATACCATGCAGAAGGAGGCAAAGTAAGGAGGTGGCTTTAGGTCAGCCAGCATTTTCCTGACA CCAGGGACCAGGCTGCCTTCCCACTAGCTGTATTGTTTA;
    • Chr17: 37880969-37881082 (Hg19) amplicon of the ERBB2 gene, the sequence of which is shown in SEQ ID:12:CATACCCTCTCAGCGTACCCTTGTCCCCAGGAAGCATACGTGATGGCTGGTGTGGGCTCCC CATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATG CCCTATGGCTGCCTCTTAGACCATGTCCG;
    • Chr12: 25380261-25380363 (Hg19) amplicon of the KRAS gene, the sequence of which is shown in SEQ ID:13 :AGTCCTCATGTACTGGTCCCTCATTGCACTGTACTCCTCTTGACCTGCTGTGTCGAGAATAT CCAAGAGACAGGTTTCTCCATCAATTACTACTTGCTTCCTGTAGGAATCCTGAGAAGGGAGAAAC ACAGTCTGGATTATTACAGTGCA;
    • Chr12: 25398183-25398310 (Hg19) amplicon of the KRAS gene, the sequence of which is shown in SEQ ID:14:AAAGAATGGTCCTGCACCAGTAATATGCATATTAAAACAAGATTTACCTCTATTGTTGGAT CATATTCGTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCACCA GCTCCAACTACCACAAGTTTATATTCAGTCATTTTCAGCAGGCCTT;
    • Chr7: 116340233-116340335 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:15 :TCGATCTGCCATGTGTGCATTCCCTATCAAATATGTCAACGACTTCTTCAACAAGATCGTCA ACAAAAACAATGTGAGATGTCTCCAGCATTTTTACGGACCCAATCATGAGCACTGCTTTAATAGG GTAAGTCACATCAGTTCCC;
    • Chr7: 116411880-116412005 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:16:CCATGATAGCCGTCTTTAACAAGCTCTTTCTTTCTCTCTGTTTTAAGATCTGGGCAGTGAAT TAGTTCGCTACGATGCAAGAGTACACACTCCTCATTTGGATAGGCTTGTAAGTGCCCGAAGTGTA AGCCCAACTACAGAAATGGTTTCAAATGAATCTGTAGACTACCGAGCT;
    • Chr7: 116417426-116417546 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:17:ATGTTACGCAGTGCTAACCAAGTTCTTTCTTTTGCACAGGGCATTTTGGTTGTGTATATCAT GGGACTTTGTTGGACAATGATGGCAAGAAAATTCACTGTGCTGTGAAATCCTTGAACAGTAAGTG GCATTTTATTTAACCATGGAGTATACTTTTGTGGTTTGCAAC;
    • Chr7: 116423399-116423499 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:18:CAGTCAAGGTTGCTGATTTTGGTCTTGCCAGAGACATGTATGATAAAGAATACTATAGTGT ACACAACAAAACAGGTGCAAAGCTGCCAGTGAAGTGGATGGCTTTGGAAAGTCTGCAAACTCAA AAGTTTACCACCAAGTCAGATGTG;
    • Chr1: 115256507-115256586 (Hg19) amplicon of the NRAS gene, the sequence of which is shown in SEQ ID:19 :TTCGCCTGTCCTCATGTATTGGTCTCTCATGGCACTGTACTCTTCTTGTCCAGCTGTATCCAG TATGTCCAACAAACAGGTTTCACCATCTATAACCACTTGTTTTCTGTAAGAATCCTGGGGGTG;
    • Chr1: 115258651-115258755 (Hg19) amplicon of the NRAS gene, the sequence of which is shown in SEQ ID:20:TGAGAGACAGGATCAGGTCAGCGGGCTACCACTGGGCCTCACCTCTATGGTGGGATCATAT TCATCTACAAAGTGGTTCTGGATTAGCTGGATTGTCAGTGCGCTTTTCCCAACACCACCTGCTCCA ACCACCACCAGTTTGTACTCAG;
    • Chr3: 178936056-178936179 (Hg19) amplicon of the PIK3CA gene, the sequence of which is shown in SEQ ID:21:GGAAAATGACAAAGAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACT GAGCAGGAGAAAGATTTTCTATGGAGTCACAGGTAAGTGCTAAAATGGAGATTCTCTGTTTCTTT TTCTTTATTACAGAAAAAATAACTGAATTTGGCTGATCTCAGCATGTT;
    • Chr3: 178952000-178952092 (Hg19) amplicon of the PIK3CA gene, the sequence of which is shown in SEQ ID:22:ATGCCAGAACTACAATCTTTTGATGACATTGCATACATTCGAAAGACCCTAGCCTTAGATA AAACTGAGCAAGAGGCTTTGGAGTATTTCATGAAACAAATGAATGATGCACATCATGGTGGCTG GACAACAAAAATGGATTG;
    • Chr17: 7577027-7577154 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:23:CTTCTTGTCCTGCTTGCTTACCTCGCTTAGTGCTCCCTGGGGGCAGCTCGTGGTGAGGCTCC CCTTTCTTGCGGAGATTCTCTTCCTCTGTGCGCCGGTCTCTCCCAGGACAGGCACAAACACGCACC TCAAAGCTGTTCCGTCCCAGTAGATTACCACTACTCAGGATAGGAAAAGAG;
    • Chr17: 7577507-7577613 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:24:GCAAGTGGCTCCTGACCTGGAGTCTTCCAGTGTGATGATGGTGAGGATGGGCCTCCGGTTC ATGCCGCCCATGCAGGAACTGTTACACATGTAGTTGTAGTGGATGGTGGTACAGTCAGAGCCAAC CTAGGAGATAACACAGGCCCAAGA;
    • Chr17: 7578182-7578298 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:25:CCCCAGTTGCAAACCAGACCTCAGGCGGCTCATAGGGCACCACCACACTATGTCGAAAAGT GTTTCTGTCATCCAAATACTCCACACGCAAATTTCCTTCCACTCGGATAAGATGCTGAGGAGGGG CCAGACCTAAGAGCAATCAGTGAGGAATCAGAGG;
    • Chr17: 7578389-7578537 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:26:ACCATCGCTATCTGAGCAGCGCTCATGGTGGGGGCAGCGCCTCACAACCTCCGTCATGTGC TGTGACTGCTTGTAGATGGCCATGGCGCGGACGCGGGTGCCGGGCGGGGGTGTGGAATCAACCC ACAGCTGCACAGGGCAGGTCTTGGCCAGTTGGCAAAACATCTTGTTGAGGGCAGGGGAGTACTG.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr2:29432588-29432707 (Hg19) amplicon of the ALK gene is shown as SEQ ID: 27: ACTGCCTCTTGACCTGTCC; the specific downstream primer sequence designed according to the Chr2:29432588-29432707 (Hg19) amplicon of the ALK gene is shown as SEQ ID: 28: TAAGGGACAAGCAGCCACAC.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr2: 29443616-29443730 (Hg19) amplicon of ALK gene is shown as SEQ ID: 29: CCAGACTCAGCTCAGTTAATTTTGG; the specific downstream primer sequence designed according to the Chr2: 29443616-29443730 (Hg19) amplicon of the ALK gene is shown as SEQ ID: 30: CGGAGGAAGGACTTGAGGT.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr7: 140453091-140453197(Hg19) amplicon of BRAF gene is shown as SEQ ID: 31: CTACTGTTTTCCTTTACTTACTACACCTC; the specific downstream primer sequence designed according to the Chr7: 140453091-140453197(Hg19) amplicon of the BRAF gene is shown as SEQ ID: 32: CCTCAATTCTTACCATCCACAAAATGG.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr7: 55241604-55241726(Hg19) amplicon of EGFR gene is shown as SEQ ID: 33: TGACCCTTGTCTCTGTGTTCTTG; the specific downstream primer sequence designed according to the Chr7: 55241604-55241726(Hg19) amplicon of the BRAF gene is shown as SEQ ID: 34: CCAGGGACCTTACCTTATACACC.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr7: 55242398-55242513 (Hg19) amplicon of EGFR gene is shown as SEQ ID:35: ACAATTGCCAGTTAACGTCTTCC; the specific downstream primer sequence designed according to the Chr7: 55242398-55242513 (Hg19) amplicon of the EGFR gene is shown as SEQ ID: 36: ACACAGCAAAGCAGAAACTCAC.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr7: 55248970-55249096 (Hg19) amplicon of EGFR gene is shown as SEQ ID: 37: GAAGCCACACTGACGTGC; the specific downstream primer sequence designed according to the Chr7: 55248970-55249096 (Hg19) amplicon of the EGFR gene is shown as SEQ ID: 38: GTGTTCCCGGACATAGTCCAG.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr7: 55259505-55259621 (Hg19) amplicon of EGFR gene is shown as SEQ ID: 39: CCGCAGCATGTCAAGATCACA; the specific downstream primer sequence designed according to the Chr7: 55259505-55259621 (Hg19) amplicon of the EGFR gene is shown as SEQ ID: 40: TAAACAATACAGCTAGTGGGAAGGC.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr17: 37880969-37881082 (Hg19) amplicon of ERBB2 gene is shown as SEQ ID: 41: CATACCCTCTCAGCGTACCC; the specific downstream primer sequence designed according to the Chr17: 37880969-37881082 (Hg19) amplicon of the ERBB2 gene is shown as SEQ ID: 42: CGGACATGGTCTAAGAGGCAG.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr12: 25380261-25380363 (Hg19) amplicon of KRAS gene is shown as SEQ ID: 43: TGCACTGTAATAATCCAGACTGTGT; the specific downstream primer sequence designed according to the Chr12: 25380261-25380363 (Hg19) amplicon of the KRAS gene is shown as SEQ ID: 44: AGTCCTCATGTACTGGTCCCTC.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr12: 25398183-25398310 (Hg19) amplicon of KRAS gene is shown as SEQ ID: 45: AAGGCCTGCTGAAAATGACTGA; the specific downstream primer sequence designed according to the Chr12: 25398183-25398310 (Hg19) amplicon of the KRAS gene is shown as SEQ ID: 46: AAAGAATGGTCCTGCACCAGTA.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr7: 116340233-116340335 (Hg19) amplicon of MET gene is shown as SEQ ID: 47: TCGATCTGCCATGTGTGCATT; the specific downstream primer sequence designed according to the Chr7: 116340233-116340335 (Hg19) amplicon of the MET gene is shown as SEQ ID: 48: GGGAACTGATGTGACTTACCCT.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr7: 116411880-116412005 (Hg19) amplicon of MET gene is shown as SEQ ID: 49: CCATGATAGCCGTCTTTAACAAGC; the specific downstream primer sequence designed according to the Chr7: 116411880-116412005 (Hg19) amplicon of the MET gene is shown as SEQ ID: 50: AGCTCGGTAGTCTACAGATTCATTT.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr7: 116417426-116417546 (Hg19) amplicon of MET gene is shown as SEQ ID: 51: ATGTTACGCAGTGCTAACCAAG; the specific downstream primer sequence designed according to the Chr7: 116417426-116417546 (Hg19) amplicon of the MET gene is shown as SEQ ID: 52: GTTGCAAACCACAAAAGTATACTCCA.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr7: 116423399-116423499 (Hg19) amplicon of MET gene is shown as SEQ ID: 53: CAGTCAAGGTTGCTGATTTTGGTC; the specific downstream primer sequence designed according to the Chr7: 116423399-116423499 (Hg19) amplicon of the MET gene is shown as SEQ ID: 54: CACATCTGACTTGGTGGTAAACTT.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr1: 115256507-115256586 (Hg19) amplicon of NRAS gene is shown as SEQ ID: 55: CACCCCCAGGATTCTTACAGAAAA; the specific downstream primer sequence designed according to the Chr1: 115256507-115256586 (Hg19) amplicon of the NRAS gene is shown as SEQ ID: 56: TTCGCCTGTCCTCATGTATTGG.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr1: 115258651-115258755 (Hg19) amplicon of NRAS gene is shown as SEQ ID: 57: CTGAGTACAAACTGGTGGTGGT; the specific downstream primer sequence designed according to the Chr1: 115258651-115258755 (Hg19) amplicon of the NRAS gene is shown as SEQ ID: 58: TGAGAGACAGGATCAGGTCAGC.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr3: 178936056-178936179 (Hg19) amplicon of PIK3CA gene is shown as SEQ ID: 59: GGAAAATGACAAAGAACAGCTCAAAG; the specific downstream primer sequence designed according to the Chr3: 178936056-178936179 (Hg19) amplicon of the PIK3CA gene is shown as SEQ ID: 60: AACATGCTGAGATCAGCCAAATTC.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr3: 178952000-178952092 (Hg19) amplicon of PIK3CA gene is shown as SEQ ID: 61: ATGCCAGAACTACAATCTTTTGATGAC; the specific downstream primer sequence designed according to the Chr3: 178952000-178952092 (Hg19) amplicon of the PIK3CA gene is shown as SEQ ID: 62: CAATCCATTTTTGTTGTCCAGCC.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr17: 7577027-7577154 (Hg19) amplicon of TP53 gene is shown as SEQ ID: 63: CTCTTTTCCTATCCTGAGTAGTGGTAATC; the specific downstream primer sequence designed according to the Chr17: 7577027-7577154 (Hg19) amplicon of the TP53 gene is shown as SEQ ID: 64: CTTCTTGTCCTGCTTGCTTACC.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr17: 7577507-7577613 (Hg19) amplicon of TP53 gene is shown as SEQ ID: 65: TCTTGGGCCTGTGTTATCTCCTAG; the specific downstream primer sequence designed according to the Chr17: 7577507-7577613 (Hg19) amplicon of the TP53 gene is shown as SEQ ID: 66: GCAAGTGGCTCCTGACCTG.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr17: 7578182-7578298 (Hg19) amplicon of TP53 gene is shown as SEQ ID: 67: CCTCTGATTCCTCACTGATTGCTC; the specific downstream primer sequence designed according to the Chr17: 7578182-7578298 (Hg19) amplicon of the TP53 gene is shown as SEQ ID: 68: CCCCAGTTGCAAACCAGAC.
  • In an embodiment of the present invention, the specific upstream primer sequence designed according to the Chr17: 7578389-7578537 (Hg19) amplicon of TP53 gene is shown as SEQ ID: 69: CAGTACTCCCCTGCCCTCAA; the specific downstream primer sequence designed according to the Chr17: 7578389-7578537 (Hg19) amplicon of the TP53 gene is shown as SEQ ID: 70: ACCATCGCTATCTGAGCAGC.
  • In an embodiment of the present invention, the target amplicons are the following 22 species:
    • Chr2:29432588-29432707 (Hg19) amplicon of the ALK gene, the sequence of which is shown in SEQ ID:5;
    • Chr2: 29443616-29443730 (Hg19) amplicon of the ALK gene, the sequence of which is shown in SEQ ID:6;
    • Chr7: 140453091-140453197(Hg19) amplicon of the BRAF gene, the sequence of which is shown in SEQ ID:7;
    • Chr7: 55241604-55241726(Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:8;
    • Chr7: 55242398-55242513 (Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:9;
    • Chr7: 55248970-55249096 (Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:10;
    • Chr7: 55259505-55259621 (Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:11;
    • Chr17: 37880969-37881082 (Hg19) amplicon of the ERBB2 gene, the sequence of which is shown in SEQ ID:12;
    • Chr12: 25380261-25380363 (Hg19) amplicon of the KRAS gene, the sequence of which is shown in SEQ ID:13;
    • Chr12: 25398183-25398310 (Hg19) amplicon of the KRAS gene, the sequence of which is shown in SEQ ID:14;
    • Chr7: 116340233-116340335 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:15;
    • Chr7: 116411880-116412005 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:16;
    • Chr7: 116417426-116417546 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:17;
    • Chr7: 116423399-116423499 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:18;
    • Chr1: 115256507-115256586 (Hg19) amplicon of the NRAS gene, the sequence of which is shown in SEQ ID:19;
    • Chr1: 115258651-115258755 (Hg19) amplicon of the NRAS gene, the sequence of which is shown in SEQ ID:20;
    • Chr3: 178936056-178936179 (Hg19) amplicon of the PIK3CA gene, the sequence of which is shown in SEQ ID:21;
    • Chr3: 178952000-178952092 (Hg19) amplicon of the PIK3CA gene, the sequence of which is shown in SEQ ID:22;
    • Chr17: 7577027-7577154 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:23;
    • Chr17: 7577507-7577613 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:24;
    • Chr17: 7578182-7578298 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:25; and
    • Chr17: 7578389-7578537 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:26.
  • In an embodiment of the present invention, the molar ratio of the combination of the upstream fusion primers designed according to the above 22 target amplicons, the combination of the downstream fusion primers designed according to the above 22 target amplicons, the upstream universal primer and the downstream universal primer is: 0.1-0.3: 0.1-0.3: 0.5-1: 0.5-1, for example, 0.1:0.1:0.5:0.5.
  • In an embodiment of the present invention, the molar ratio of the upstream fusion primer designed according to the Chr2:29432588-29432707 (Hg19) amplicon of ALK gene, the upstream fusion primer designed according to the Chr2: 29443616-29443730 (Hg19) amplicon of ALK gene, the upstream fusion primer designed according to the Chr7: 140453091-140453197(Hg19) amplicon of BRAF gene, the upstream fusion primer designed according to the Chr7: 55241604-55241726(Hg19) amplicon of EGFR gene, the upstream fusion primer designed according to the Chr7: 55242398-55242513 (Hg19) amplicon of EGFR gene, the upstream fusion primer designed according to the Chr7: 55248970-55249096 (Hg19) amplicon of EGFR gene, the upstream fusion primer designed according to the Chr7: 55259505-55259621 (Hg19) amplicon of EGFR gene, the upstream fusion primer designed according to the Chr17: 37880969-37881082 (Hg19) amplicon of ERBB2 gene; the upstream fusion primer designed according to the Chr12: 25380261-25380363 (Hg19) amplicon of KRAS gene, the upstream fusion primer designed according to the Chr12: 25398183-25398310 (Hg19) amplicon of KRAS gene ; the upstream fusion primer designed according to the Chr7: 116340233-116340335 (Hg19) amplicon of MET gene, the upstream fusion primer designed according to the Chr7: 116411880-116412005 (Hg19) amplicon of MET gene, the upstream fusion primer designed according to the Chr7: 116417426-116417546 (Hg19) amplicon of MET gene, the upstream fusion primer designed according to the Chr7: 116423399-116423499 (Hg19) amplicon of MET gene, the upstream fusion primer designed according to the Chr1: 115256507-115256586 (Hg19) amplicon of NRAS gene, the upstream fusion primer designed according to the Chr1: 115258651-115258755 (Hg19) amplicon of NRAS gene, the upstream fusion primer designed according to the Chr3: 178936056-178936179 (Hg19) amplicon of PIK3CA gene, the upstream fusion primer designed according to the Chr3: 178952000-178952092 (Hg19) amplicon of PIK3CA gene, the upstream fusion primer designed according to the Chr17: 7577027-7577154 (Hg19) amplicon of TP53 gene, the upstream fusion primer designed according to the Chr17: 7577507-7577613 (Hg19) amplicon of TP53 gene, the upstream fusion primer designed according to the Chr17: 7578182-7578298 (Hg19) amplicon of TP53 gene, and the upstream fusion primer designed according to the Chr17: 7578389-7578537 (Hg19) amplicon of TP53 gene is: 1:2:1:4:2:1:2:4:2:2:2:2:1:4:2:2:2:2:4:2:4:2.
  • In an embodiment of the present invention, the PCR reaction system includes the following components:
    PCR master mix 10µl;
    DNA sample 1-8µl total 20 ng;
    Primer combination for constructing an amplicon library of the same DNA sample 2µl;
    DNAase-free H2O making up to 20µl.
  • In an embodiment of the present invention, the PCR master mix is KAPA HiFi PCR Kits 2×.
  • In an embodiment of the present invention, the reaction procedure for performing PCR is:
    Temperature Time Number of cycles
    98°C 30s
    98°C 10s 22 cycles
    60°C 90s
    72°C 90s
    72°C 10min
    4°C ----
  • In an embodiment of the present invention, after the PCR reaction, a step of purifying the PCR amplification product is also included.
  • Compared with the prior art, the present invention has the following advantages:
    The method disclosed in the present invention is based on the design of the PGM platform, and can effectively amplify multiple target regions (amplicons) at the same time. In the process of using the library for construction, the present invention only involves one round of PCR reaction and one round of product purification steps, which greatly simplifies the experimental operation of the existing commercial kit (such as PCR process, purification step, digestion and joints, etc.) Step), and saves the construction time. The entire database construction process only takes 2.5 hours (including the same sample of DNA and RNA database).
  • Effectively elimination of sample and library contamination is achieved. The significant simplified operation process makes the library construction process more secure and reliable, and the reduction of operation process and steps effectively eliminates the library pollution that may be caused during the database construction process.
  • Streamlined bioinformatics analysis process is obtained. The amplicon library obtained by the method has a single structure and reliable data, and the DNA strand composition of the obtained library is simple and clear, and the subsequent bioinformatics analysis is more simplified.
  • After the library is constructed, the library is only needed to be quantified by the instrument "Qubit 2.0", which eliminates quantification step by the instrument "qPCR". Therefore, the database construction time is shortened and corresponding operation steps are reduced, and the experimental errors that may be caused by the cumbersome experimental process are avoided.
    • BRIEF DESCRIPTION OF THE DRAWINGS
    • Figure 1 is a distribution diagram of an amplification product detected after completion of construction of an amplicon library in Example 1 of the present invention.
    • Figure 2 is a related parameter of 22 amplicons in the library obtained in Example 1 of the present invention.
    DETAILED DESCRIPITON OF THE EMBODIMENTS
  • The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings, but it is understood that the scope of the present invention is not limited by the specific embodiments.
    • Example 1
  • The samples to be tested are 6 FFPE samples (ie, formalin-fixed paraffin-embedded samples, FFPE stands for Formalin-Fixed and Parrffin-Embedded), 4 of which are FFPE samples from patients with non-small cell lung cancer, and 2 are non- FFPE samples from patients with cancer. Amplified primers are used to construct an amplicon DNA library from 6 FFPE samples using a specific designed fusion primer. The specific process is as follows:
    • 1. Extraction of genomic DNA:
  • The genomic DNA in the FFPE sample is extracted using the "Qiagen FFPE DNA Kit". The detailed steps for extraction can be referred to the kit instructions. The genomic DNA is dissolved in "Tris-HCl" buffer, quality of the extracted DNA is detected using "Nano Drop". After the concentration of the sample DNA is detected using the instrument "quiz 3.0", each genomic DNA sample is diluted to a concentration of 20 ng/µl.
    • 2. Design and synthesize primers:
  • An upstream fusion primer is designed according to the target amplicon. The upstream fusion primer includes a first linker sequence arranged in the order of 5' to 3' and a specific upstream primer sequence that is designed according to the target amplicon;
    A downstream fusion primer is designed according to the target amplicon. The downstream fusion primer includes a second linker sequence arranged in the order of 5' to 3' and a specific downstream primer sequence designed according to the target amplicon;
    An upstream universal primer comprises a third linker sequence, barcode sequence and a first linker sequence arranged in the order of 5' to 3'; and
    A downstream universal primer comprises a universal sequence and a second linker sequence arranged in the order of 5' to 3'.
  • In the primer combination for constructing the amplicon library of the DNA sample, the information of the specific upstream primer sequence and the specific downstream primer sequence designed according to the target amplicon are as follows:
    Information on the different target amplicons is given in the table below, and the specific upstream primer sequence "Special Primer Start" and the specific downstream primer sequence "Special Primer End" designed for these amplicons are also given. Sequences of upstream fusion primers designed according to the target amplicon, downstream fusion primers designed according to the target amplicon, upstream universal primers, and downstream universal primers are also given. Puf representes an alternative upstream universal primer and Pur representes a downstream universal primer.
    Gene/Amplicons Chr Amp Start Ins Start Ins End Amp End length Primer name Special Primer Start Primer name Special Primer End
    ALK-001 2 29432569 29432588 29432707 29432727 159 Pspf-1
    Figure imgb0001
    		 pspr-1
    Figure imgb0002
    ALK-002 2 29443591 29443616 29443730 29443749 159 Pspf-2
    Figure imgb0003
    		 pspr-2
    Figure imgb0004
    BRAF-001 7 140453062 140453091 140453197 140453224 163 Pspf-3
    Figure imgb0005
    		 pspr-3
    Figure imgb0006
    EGFR-001 7 55241581 55241604 55241726 55241749 169 Pspf-4
    Figure imgb0007
    		 pspr-4
    Figure imgb0008
    EGFR-002 7 55242375 55242398 55242513 55242535 161 Pspf-5
    Figure imgb0009
    		 pspr-5
    Figure imgb0010
    EGFR-003 7 55248952 55248970 55249096 55249117 166 Pspf-6
    Figure imgb0011
    		 pspr-6
    Figure imgb0012
    EGFR-004 7 55259484 55259505 55259621 55259646 163 Pspf-7
    Figure imgb0013
    		 pspr- 7
    Figure imgb0014
    ERBB2-001 17 37880949 37880969 37881082 37881103 155 Pspf-8
    Figure imgb0015
    		 pspr-8
    Figure imgb0016
    KRAS-001 12 25380236 25380261 25380363 25380385 150 Pspf-9
    Figure imgb0017
    		 pspr-9
    Figure imgb0018
    KRAS-002 12 25398161 25398183 25398310 25398332 172 Pspf-10
    Figure imgb0019
    		 pspr-10
    Figure imgb0020
    MET-001 7 116340212 116340233 116340335 116340357 146 Pspf-11
    Figure imgb0021
    		 pspr-11
    Figure imgb0022
    MET-002 7 116411856 116411880 116412005 116412030 175 Pspf-12
    Figure imgb0023
    		 pspr-12
    Figure imgb0024
    MET-003 7 116417404 116417426 116417546 116417572 169 Pspf-13
    Figure imgb0025
    		 pspr-13
    Figure imgb0026
    MET-004 7 116423375 116423399 116423499 116423523 149 Pspf-14
    Figure imgb0027
    		 pspr-14
    Figure imgb0028
    NRAS-001 1 115256483 115256507 115256586 115256608 126 Pspf-15
    Figure imgb0029
    		 pspr-15
    Figure imgb0030
    NRAS-002 1 115258629 115258651 115258755 115258777 149 Pspf-16
    Figure imgb0031
    		 pspr-16
    Figure imgb0032
    PIK3CA-001 3 178936030 178936056 178936179 178936203 174 Pspf-17
    Figure imgb0033
    		 pspr-17
    Figure imgb0034
    PIK3CA-002 3 178951973 178952000 178952092 178952115 143 Pspf-18
    Figure imgb0035
    		 pspr-18
    Figure imgb0036
    TP53-001 17 7576998 7577027 7577154 7577176 179 Pspf-19
    Figure imgb0037
    		 pspr-19
    Figure imgb0038
    TP53-002 17 7577483 7577507 7577613 7577632 150 Pspf-20
    Figure imgb0039
    		 pspr-20
    Figure imgb0040
    TP53-003 17 7578158 7578182 7578298 7578317 159 Pspf-21
    Figure imgb0041
    		 pspr- 21
    Figure imgb0042
    TP53-004 17 7578369 7578389 7578537 7578557 189 Pspf-22
    Figure imgb0043
    		 pspr-22
    Figure imgb0044
    Primer name Primer sequence
    Pspf-1 GGCATACGTCCTCGTCTAACTGCCTCTTGACCTGTCC
    Pspf-2 GGCATACGTCCTCGTCTACCAGACTCAGCTCAGTTAATTTTGG
    Pspf-3 GGCATACGTCCTCGTCTACTACTGTTTTCCTTTACTTACTACACCTC
    Pspf-4 GGCATACGTCCTCGTCTATGACCCTTGTCTCTGTGTTCTTG
    Pspf-5 GGCATACGTCCTCGTCTAACAATTGCCAGTTAACGTCTTCC
    Pspf-6 GGCATACGTCCTCGTCTAGAAGCCACACTGACGTGC
    Pspf-7 GGCATACGTCCTCGTCTACCGCAGCATGTCAAGATCACA
    Pspf-8 GGCATACGTCCTCGTCTACATACCCTCTCAGCGTACCC
    Pspf-9 GGCATACGTCCTCGTCTATGCACTGTAATAATCCAGACTGTGT
    Pspf-10 GGCATACGTCCTCGTCTAAAGGCCTGCTGAAAATGACTGA
    Pspf-11 GGCATACGTCCTCGTCTATCGATCTGCCATGTGTGCATT
    Pspf-12 GGCATACGTCCTCGTCTACCATGATAGCCGTCTTTAACAAGC
    Pspf-13 GGCATACGTCCTCGTCTAATGTTACGCAGTGCTAACCAAG
    Pspf-14 GGCATACGTCCTCGTCTACAGTCAAGGTTGCTGATTTTGGTC
    Pspf-15 GGCATACGTCCTCGTCTACACCCCCAGGATTCTTACAGAAAA
    Pspf-16 GGCATACGTCCTCGTCTACTGAGTACAAACTGGTGGTGGT
    Pspf-17 GGCATACGTCCTCGTCTAGGAAAATGACAAAGAACAGCTCAAAG
    Pspf-18 GGCATACGTCCTCGTCTAATGCCAGAACTACAATCTTTTGATGAC
    Pspf-19 GGCATACGTCCTCGTCTACTCTTTTCCTATCCTGAGTAGTGGTAATC
    Pspf-20 GGCATACGTCCTCGTCTATCTTGGGCCTGTGTTATCTCCTAG
    Pspf-21 GGCATACGTCCTCGTCTACCTCTGATTCCTCACTGATTGCTC
    Pspf-22 GGCATACGTCCTCGTCTACAGTACTCCCCTGCCCTCAA
    pspr-1 TCTATGGGCAGTCGGTGATTAAGGGACAAGCAGCCACAC
    pspr-2 TCTATGGGCAGTCGGTGATCGGAGGAAGGACTTGAGGT
    pspr-3 TCTATGGGCAGTCGGTGATCCTCAATTCTTACCATCCACAAAATGG
    pspr-4 TCT A TGGGCAGTCGGTGA TCCAGGGACCTT ACCTT A T ACACC
    pspr-5 TCTATGGGCAGTCGGTGATACACAGCAAAGCAGAAACTCAC
    pspr-6 TCTATGGGCAGTCGGTGATGTGTTCCCGGACATAGTCCAG
    pspr-7 TCTATGGGCAGTCGGTGATTAAACAATACAGCTAGTGGGAAGGC
    pspr-8 TCTATGGGCAGTCGGTGATCGGACATGGTCTAAGAGGCAG
    pspr-9 TCT A TGGGCAGTCGGTGA T AGTCCTCA TGT ACTGGTCCCTC
    pspr-10 TCTATGGGCAGTCGGTGATAAAGAATGGTCCTGCACCAGTA
    pspr-11 TCTATGGGCAGTCGGTGATGGGAACTGATGTGACTTACCCT
    pspr-12 TCTATGGGCAGTCGGTGATAGCTCGGTAGTCTACAGATTCATTT
    pspr-13 TCTATGGGCAGTCGGTGATGTTGCAAACCACAAAAGTATACTCCA
    pspr-14 TCTATGGGCAGTCGGTGATCACATCTGACTTGGTGGTAAACTT
    pspr-15 TCTATGGGCAGTCGGTGATTTCGCCTGTCCTCATGTATTGG
    pspr-16 TCTATGGGCAGTCGGTGATTGAGAGACAGGATCAGGTCAGC
    pspr-17 TCTATGGGCAGTCGGTGATAACATGCTGAGATCAGCCAAATTC
    pspr-18 TCTATGGGCAGTCGGTGATCAATCCATTTTTGTTGTCCAGCC
    pspr-19 TCTATGGGCAGTCGGTGATCTTCTTGTCCTGCTTGCTTACC
    pspr-20 TCTATGGGCAGTCGGTGATGCAAGTGGCTCCTGACCTG
    pspr-21 TCTATGGGCAGTCGGTGATCCCCAGTTGCAAACCAGAC
    pspr-22 TCTATGGGCAGTCGGTGATACCATCGCTATCTGAGCAGC
    puf-1 CCATCTCATCCCTGCGTGTCTCCGACTCAGCTTGACACCGCGGCATACGTCCTCGTCTA
    puf-2 CCATCTCATCCCTGCGTGTCTCCGACTCAGTTGGAGGCCAGCGGCATACGTCCTCGTCTA
    puf-3 CCATCTCATCCCTGCGTGTCTCCGACTCAGTGGAGCTTCCTCGGCATACGTCCTCGTCTA
    puf-4 CCATCTCATCCCTGCGTGTCTCCGACTCAGTCAGTCCGAACGGCATACGTCCTCGTCTA
    puf-5 CCATCTCATCCCTGCGTGTCTCCGACTCAGTAAGGCAACCACGGCATACGTCCTCGTCTA
    puf-6 CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCTAAGAGACGGCATACGTCCTCGTCTA
    puf-7 CCATCTCATCCCTGCGTGTCTCCGACTCAGTCCTAACATAACGGCATACGTCCTCGTCTA
    puf-8 CCATCTCATCCCTGCGTGTCTCCGACTCAGCGGACAATGGCGGCATACGTCCTCGTCTA
    puf-9 CCATCTCATCCCTGCGTGTCTCCGACTCAGTTGAGCCTATTCGGCATACGTCCTCGTCTA
    puf-10 CCATCTCATCCCTGCGTGTCTCCGACTCAGCCGCATGGAACGGCATACGTCCTCGTCTA
    pur CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT
  • The first linker sequence is GGCATACGTCCTCGTCTA, the second linker sequence is TCTATGGGCAGTCGGTGAT, the third linker sequence is CCATCTCATCCCTGCGTGTCTCCGACTCAG, and the universal sequence is CCACTACGCCTCCGCTTTCCTC.
    • 3. Form a PCR reaction system. The specific PCR reaction system is as follows:
  • PCR reaction system component content
    KAPA HiFi PCR Kits 2× 10µl
    Genomic DNA (10 ng/µl itself) 2µl
    Primer combination for constructing an amplicon library of the same DNA sample 2µl
    DNAase free H2O make up to the total of 20µl
    Total 20µl
  • Primer combinations for constructing an amplicon library of the same DNA sample are prepared by the following methods: (1) the upstream universal primer, the downstream universal primer, and each upstream fusion primer designed according to the 22 target amplicons and each downstream fusion primer are dissolved in water to a concentration of 100 µM; (2) 22 upstream fusion primers with a serial number ranging from small to large are respectively mixed with a concentration of 100µM, and the molar ratio is 1:2:1:4:2:1:2:4:2:2:2:2:2:4:2:2:2:2:4:2:4:2, so as to obtain the upstream fusion primer combination, and 22 downstream fusion primers with a concentration of 100 µM are respectively mixed with the corresponding upstream fusion primers in equal volume to obtain a downstream fusion primer combination, and then the upstream fusion primer combination and the downstream fusion primer combination are mixed in equal volume; (3) mixing in equal volume of upstream universal primers and downstream universal primers with concentrations of 100 µM; (4) the upstream fusion primer combination, the downstream fusion primer combination, the upstream universal primer and the downstream universal primer are mixed according to a molar ratio of 0.1:0.1:0.5:0.5, so that the amplicons for constructing the DNA sample are obtained. Six different sets of samples to be tested need to correspond to primer combinations containing six different barcode sequence tags.
    • 4. Carry out the PCR program. The PCR instrument is the 2720 Thermal Cycler of Applied Bio-system. The PCR reaction procedure is as follows:
  • Temperature Time Number of cycles
    98°C 30s
    98°C 10s 22 Cycles
    60°C 90s
    72°C 90s
    72°C 10min
    4°C ----
    • 5. After the PCR reaction, purification is carried out using "Agencourt AMPure XP Kit" (Cat. No. A63880/A63881/A63882) from Beckman Coulter company. The steps are as follows:
    1. 1) take out the Agencourt AMPure XP Kit 30 minutes in advance, rotate the magnetic beads in the Kit thoroughly, and keep the EP tube at room temperature.
    2. 2) After the completion of the PCR reaction, the magnetic beads are rotated again sufficiently, and 20 ul of magnetic beads are added to the system, repeatedly blow 5 times or more, or rotate thoroughly, and allow the Kit to be placed at room temperature for 5 minutes.
    3. 3) Transfer the EP tube to the magnetic stand and keep for 5 minutes until the solution is clarified. Carefully remove the supernatant with a pipette, taking care not to touch the beads.
    4. 4) Add 100 ul of freshly prepared 80% ethanol solution to each tube, and place the EP tube on the magnetic stand and rotate two turns, keep it for 5 minutes, and discard the supernatant.
    5. 5) Repeat step 4) once.
    6. 6) Open the EP tube and keep it at room temperature to make the liquid volatilize completely. Make sure the surfaces of the magnetic beads are dull, and be careful not to over-dry the magnetic beads.
    7. 7) Remove the EP tube from the magnetic stand, add 30 ul of PCR-grade purified water, rotate and mix, and keep it for 10 minutes at room temperature.
    8. 8) Place the EP tube on the magnetic stand for 2 minutes or until the solution is clarified. Carefully suck the supernatant from the side away from the magnet with a pipette, taking care not to touch the beads.
  • At this point, the amplicon library is constructed. Figure 1 shows the distribution of amplified products detected by Agilent 2200 TapeStation Systems after the completion of the library. The abscissa is the length of the fragment, the ordinate is the signal intensity (FU), and the lower peak is the 25 bp position marker, the upper peak is a 1500 bp position marker. As shown in Figure 1, the PCR products obtained by PCR amplification are concentrated in the range of 241-271 bp. Figure 1 shows that the experimental results are consistent with the experimental design. From Figure 1, the size of the constructed library and the library concentration can be judged.
    • 6. On-machine sequencing and results analysis
  • The amplicon library is obtained by the fusion primer one-step method. The amplicon sequencing is performed using the chip 318 of the Ion PGM platform, and the data amount of each library is 50 M bps. The average sequencing depth of each sample is not less than 1600X, and the single amplicon sequencing depth reached 600X. The obtained sequencing results are shown in Fig. 2. From Fig. 2, it is possible to further analyze whether or not each amplicon of the 22 amplicons is amplified and the amplification uniformity of each amplicon.
  • The results of sequencing are analyzed by data processing and bioinformatics analysis to obtain mutations in the detected genes. The data processing process includes conversion, quality control and sequence alignment of the sequencing data (reference genome is NCBI GRCh37/Hg19), mutation site analysis and other processes, and the mutation information of the detected samples is obtained through data processing analysis.
  • The actual sample collection is as follows: Among the FFPE samples of 6 subjects, no tumor-related mutations are detected in 2 normal human samples, among the 4 FFPE samples of tumor patients, p.R248W mutation is detected in Sample1, p.T790M mutation is detected in sample2, p.G12A mutation is detected in sample3, and p.E545K mutation is detected in Sample4. This result is consistent with the results of the sanger test. The practical applicability and good specificity of the present invention are fully illustrated.
  • The foregoing descriptions of specific embodiments of the present invention are presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments are chosen and described in order to explain certain principles of the invention and their practical application, to thereby enable others skilled in the art to make and utilize various exemplary embodiments of the present invention, as well as various alternatives and modifications thereof. The invention is to be limited only by the claims provided below and equivalents thereof.
    Figure imgb0045
    Figure imgb0046
    Figure imgb0047
    Figure imgb0048
    Figure imgb0049
    Figure imgb0050
    Figure imgb0051
    Figure imgb0052
    Figure imgb0053
    Figure imgb0054
    Figure imgb0055
    Figure imgb0056

Claims (10)

  1. A method for constructing an amplicon library of a DNA sample, comprising the following steps:
    1) synthesizing a primer combination which is used for constructing an amplicon library of a DNA sample, wherein the primer combination of the amplicon library used to construct the DNA sample includes:
    an upstream fusion primer designed according to a target amplicon, the upstream fusion primer comprising a first linker sequence (Bridge sequence) arranged in the order of 5' to 3' and a specific upstream primer sequence designed according to the target amplicon;
    a downstream fusion primer designed according to the target amplicon, the downstream fusion primer comprising a second linker sequence (trP1 sequence) arranged in the order of 5' to 3' and a specific downstream primer sequence designed according to the target amplicon;
    an upstream universal primer comprising a third linker sequence (A sequence), a barcode sequence and a first linker sequence arranged in the order of 5' to 3'; and
    a downstream universal primer comprising a universal sequence (Uni sequence) and a second linker sequence arranged in the order of 5' to 3';
    2) constructing a PCR reaction system for the DNA sample, and mixing the upstream fusion primers designed according to the target amplicon, the downstream fusion primers designed according to the target amplicon, the upstream universal primers and the downstream universal primers together, to serve as a primer combination in the PCR reaction system; and
    3) performing PCR.
  2. The method for constructing an amplicon library of a DNA sample according to claim 1, wherein the first linker sequence comprises a sequence of SEQ ID: 1; optionally, the second linker sequence comprises a sequence of SEQ ID 2; optionally, the third linker sequence comprises a sequence of SEQ ID: 3; optionally, the universal sequence comprises a sequence of SEQ ID: 4.
  3. The method for constructing an amplicon library of a DNA sample according to claim 1, wherein in a primer combination for constructing a plurality of amplicon libraries of the same DNA sample, the barcode sequences in the upstream universal primers are the same; in a primer combination for constructing amplicon libraries of different DNA samples, the barcode sequences in the upstream universal primers are different.
  4. The method for constructing an amplicon library of a DNA sample according to claim 1, wherein when the number of target amplicon in a same PCR reaction is greater than one, the upstream fusion primer designed according to the target amplicon is a combination of upstream fusion primers designed according to each target amplicon, the downstream fusion primer designed according to the target amplicon is a combination of downstream fusion primers designed according to each target amplicon.
  5. The method for constructing an amplicon library of a DNA sample according to claim 1, wherein the DNA sample is genomic DNA, optionally, the genomic DNA is extracted from a tissue sample or a formalin-fixed paraffin-embedded sample.
  6. The method for constructing an amplicon library of a DNA sample according to claim 1, wherein the target amplicon comprises at least one selected from the group consisting of 22 target amplicons:
    Chr2:29432588-29432707 (Hg19) amplicon of the ALK gene, the sequence of which is shown in SEQ ID:5;
    Chr2: 29443616-29443730 (Hg19) amplicon of the ALK gene, the sequence of which is shown in SEQ ID:6;
    Chr7: 140453091-140453197(Hg19) amplicon of the BRAF gene, the sequence of which is shown in SEQ ID:7;
    Chr7: 55241604-55241726(Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:8;
    Chr7: 55242398-55242513 (Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:9;
    Chr7: 55248970-55249096 (Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:10;
    Chr7: 55259505-55259621 (Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:11;
    Chr17: 37880969-37881082 (Hg19) amplicon of the ERBB2 gene, the sequence of which is shown in SEQ ID:12;
    Chr12: 25380261-25380363 (Hg19) amplicon of the KRAS gene, the sequence of which is shown in SEQ ID:13;
    Chr12: 25398183-25398310 (Hg19) amplicon of the KRAS gene, the sequence of which is shown in SEQ ID:14;
    Chr7: 116340233-116340335 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:15;
    Chr7: 116411880-116412005 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:16;
    Chr7: 116417426-116417546 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:17;
    Chr7: 116423399-116423499 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:18;
    Chr1: 115256507-115256586 (Hg19) amplicon of the NRAS gene, the sequence of which is shown in SEQ ID:19;
    Chr1: 115258651-115258755 (Hg19) amplicon of the NRAS gene, the sequence of which is shown in SEQ ID:20;
    Chr3: 178936056-178936179 (Hg19) amplicon of the PIK3CA gene, the sequence of which is shown in SEQ ID:21;
    Chr3: 178952000-178952092 (Hg19) amplicon of the PIK3CA gene, the sequence of which is shown in SEQ ID:22;
    Chr17: 7577027-7577154 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:23;
    Chr17: 7577507-7577613 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:24;
    Chr17: 7578182-7578298 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:25; and
    Chr17: 7578389-7578537 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:26.
  7. The method for constructing an amplicon library of a DNA sample according to claim 6, wherein,
    the specific upstream primer sequence designed according to the Chr2:29432588-29432707 (Hg19) amplicon of the ALK gene is shown as SEQ ID: 27; the specific downstream primer sequence designed according to the Chr2:29432588-29432707 (Hg19) amplicon of the ALK gene is shown as SEQ ID: 28;
    optionally, the specific upstream primer sequence designed according to the Chr2: 29443616-29443730 (Hg19) amplicon of ALK gene is shown as SEQ ID: 29; the specific downstream primer sequence designed according to the Chr2: 29443616-29443730 (Hg19) amplicon of the ALK gene is shown as SEQ ID: 30;
    optionally, the specific upstream primer sequence designed according to the Chr7: 140453091-140453197(Hg19) amplicon of BRAF gene is shown as SEQ ID: 31; the specific downstream primer sequence designed according to the Chr7: 140453091-140453197(Hg19) amplicon of the BRAF gene is shown as SEQ ID: 32;
    optionally, the specific upstream primer sequence designed according to the Chr7: 55241604-55241726(Hg19) amplicon of EGFR gene is shown as SEQ ID: 33; the specific downstream primer sequence designed according to the Chr7: 55241604-55241726(Hg19) amplicon of the BRAF gene is shown as SEQ ID: 34;
    optionally, the specific upstream primer sequence designed according to the Chr7: 55242398-55242513 (Hg19) amplicon of EGFR gene is shown as SEQ ID:35; the specific downstream primer sequence designed according to the Chr7: 55242398-55242513 (Hg19) amplicon of the EGFR gene is shown as SEQ ID: 36;
    optionally, the specific upstream primer sequence designed according to the Chr7: 55248970-55249096 (Hg19) amplicon of EGFR gene is shown as SEQ ID: 37; the specific downstream primer sequence designed according to the Chr7: 55248970-55249096 (Hg19) amplicon of the EGFR gene is shown as SEQ ID: 38;
    optionally, the specific upstream primer sequence designed according to the Chr7: 55259505-55259621 (Hg19) amplicon of EGFR gene is shown as SEQ ID: 39; the specific downstream primer sequence designed according to the Chr7: 55259505-55259621 (Hg19) amplicon of the EGFR gene is shown as SEQ ID: 40;
    optionally, the specific upstream primer sequence designed according to the Chr17: 37880969-37881082 (Hg19) amplicon of ERBB2 gene is shown as SEQ ID: 41; the specific downstream primer sequence designed according to the Chr17: 37880969-37881082 (Hg19) amplicon of the ERBB2 gene is shown as SEQ ID: 42;
    optionally, the specific upstream primer sequence designed according to the Chr12: 25380261-25380363 (Hg19) amplicon of KRAS gene is shown as SEQ ID: 43; the specific downstream primer sequence designed according to the Chr12: 25380261-25380363 (Hg19) amplicon of the KRAS gene is shown as SEQ ID: 44;
    optionally, the specific upstream primer sequence designed according to the Chr12: 25398183-25398310 (Hg19) amplicon of KRAS gene is shown as SEQ ID: 45; the specific downstream primer sequence designed according to the Chr12: 25398183-25398310 (Hg19) amplicon of the KRAS gene is shown as SEQ ID: 46;
    optionally, the specific upstream primer sequence designed according to the Chr7: 116340233-116340335 (Hg19) amplicon of MET gene is shown as SEQ ID: 47; the specific downstream primer sequence designed according to the Chr7: 116340233-116340335 (Hg19) amplicon of the MET gene is shown as SEQ ID: 48;
    optionally, the specific upstream primer sequence designed according to the Chr7: 116411880-116412005 (Hg19) amplicon of MET gene is shown as SEQ ID: 49; the specific downstream primer sequence designed according to the Chr7: 116411880-116412005 (Hg19) amplicon of the MET gene is shown as SEQ ID: 50;
    optionally, the specific upstream primer sequence designed according to the Chr7: 116417426-116417546 (Hg19) amplicon of MET gene is shown as SEQ ID: 51; the specific downstream primer sequence designed according to the Chr7: 116417426-116417546 (Hg19) amplicon of the MET gene is shown as SEQ ID: 52;
    optionally, the specific upstream primer sequence designed according to the Chr7: 116423399-116423499 (Hg19) amplicon of MET gene is shown as SEQ ID: 53; the specific downstream primer sequence designed according to the Chr7: 116423399-116423499 (Hg19) amplicon of the MET gene is shown as SEQ ID: 54;
    optionally, the specific upstream primer sequence designed according to the Chr1: 115256507-115256586 (Hg19) amplicon of NRAS gene is shown as SEQ ID: 55; the specific downstream primer sequence designed according to the Chr1: 115256507-115256586 (Hg19) amplicon of the NRAS gene is shown as SEQ ID: 56;
    optionally, the specific upstream primer sequence designed according to the Chr1: 115258651-115258755 (Hg19) amplicon of NRAS gene is shown as SEQ ID: 57; the specific downstream primer sequence designed according to the Chr1: 115258651-115258755 (Hg19) amplicon of the NRAS gene is shown as SEQ ID: 58;
    optionally, the specific upstream primer sequence designed according to the Chr3: 178936056-178936179 (Hg19) amplicon of PIK3CA gene is shown as SEQ ID: 59; the specific downstream primer sequence designed according to the Chr3: 178936056-178936179 (Hg19) amplicon of the PIK3CA gene is shown as SEQ ID: 60;
    optionally, the specific upstream primer sequence designed according to the Chr3: 178952000-178952092 (Hg19) amplicon of PIK3CA gene is shown as SEQ ID: 61; the specific downstream primer sequence designed according to the Chr3: 178952000-178952092 (Hg19) amplicon of the PIK3CA gene is shown as SEQ ID: 62;
    optionally, the specific upstream primer sequence designed according to the Chr17: 7577027-7577154 (Hg19) amplicon of TP53 gene is shown as SEQ ID: 63; the specific downstream primer sequence designed according to the Chr17: 7577027-7577154 (Hg19) amplicon of the TP53 gene is shown as SEQ ID: 64;
    optionally, the specific upstream primer sequence designed according to the Chr17: 7577507-7577613 (Hg19) amplicon of TP53 gene is shown as SEQ ID: 65; the specific downstream primer sequence designed according to the Chr17: 7577507-7577613 (Hg19) amplicon of the TP53 gene is shown as SEQ ID: 66;
    optionally, the specific upstream primer sequence designed according to the Chr17: 7578182-7578298 (Hg19) amplicon of TP53 gene is shown as SEQ ID: 67; the specific downstream primer sequence designed according to the Chr17: 7578182-7578298 (Hg19) amplicon of the TP53 gene is shown as SEQ ID: 68;
    optionally, the specific upstream primer sequence designed according to the Chr17: 7578389-7578537 (Hg19) amplicon of TP53 gene is shown as SEQ ID: 69; the specific downstream primer sequence designed according to the Chr17: 7578389-7578537 (Hg19) amplicon of the TP53 gene is shown as SEQ ID: 70.
  8. The method for constructing an amplicon library of a DNA sample according to claim 1, wherein the target amplicons are the following 22 species:
    Chr2:29432588-29432707 (Hg19) amplicon of the ALK gene, the sequence of which is shown in SEQ ID:5;
    Chr2: 29443616-29443730 (Hg19) amplicon of the ALK gene, the sequence of which is shown in SEQ ID:6;
    Chr7: 140453091-140453197(Hg19) amplicon of the BRAF gene, the sequence of which is shown in SEQ ID:7;
    Chr7: 55241604-55241726(Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:8;
    Chr7: 55242398-55242513 (Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:9;
    Chr7: 55248970-55249096 (Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:10;
    Chr7: 55259505-55259621 (Hg19) amplicon of the EGFR gene, the sequence of which is shown in SEQ ID:11;
    Chr17: 37880969-37881082 (Hg19) amplicon of the ERBB2 gene, the sequence of which is shown in SEQ ID:12;
    Chr12: 25380261-25380363 (Hg19) amplicon of the KRAS gene, the sequence of which is shown in SEQ ID:13;
    Chr12: 25398183-25398310 (Hg19) amplicon of the KRAS gene, the sequence of which is shown in SEQ ID:14;
    Chr7: 116340233-116340335 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:15;
    Chr7: 116411880-116412005 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:16;
    Chr7: 116417426-116417546 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:17;
    Chr7: 116423399-116423499 (Hg19) amplicon of the MET gene, the sequence of which is shown in SEQ ID:18;
    Chr1: 115256507-115256586 (Hg19) amplicon of the NRAS gene, the sequence of which is shown in SEQ ID:19;
    Chr1: 115258651-115258755 (Hg19) amplicon of the NRAS gene, the sequence of which is shown in SEQ ID:20;
    Chr3: 178936056-178936179 (Hg19) amplicon of the PIK3CA gene, the sequence of which is shown in SEQ ID:21;
    Chr3: 178952000-178952092 (Hg19) amplicon of the PIK3CA gene, the sequence of which is shown in SEQ ID:22;
    Chr17: 7577027-7577154 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:23;
    Chr17: 7577507-7577613 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:24;
    Chr17: 7578182-7578298 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:25; and
    Chr17: 7578389-7578537 (Hg19) amplicon of the TP53 gene, the sequence of which is shown in SEQ ID:26.
  9. The method for constructing an amplicon library of a DNA sample according to claim 8, wherein the molar ratio of the combination of the upstream fusion primers designed according to the 22 target amplicons of claim 8, the combination of the downstream fusion primers designed according to the above 22 target amplicons, the upstream universal primer and the downstream universal primer is:
    0.1-0.3: 0.1-0.3: 0.5-1: 0.5-1.
  10. The method for constructing an amplicon library of a DNA sample according to claim 9, wherein the PCR reaction system includes the following components: PCR master mix, 10µl; DNA sample, 1-8µl total 20 ng; Primer combination for constructing an amplicon library of the same DNA sample, 2µl; DNAase-free H2O, makinge up to 20µl
EP18780976.9A 2017-04-05 2018-03-28 Method for constructing amplicon library through one-step process Pending EP3608452A4 (en)

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